PROJECT SUMMARY This Supplement to parent grant 1R01 GM120574 requests funds to purchase a Helium Recovery System. Integral Membrane Proteins (IMPs) include many biomedically-important gate keepers, receptors, transporters, homeostasis regulators, and potential drug discovery targets. Three-dimensional (3D) structure determination of IMPs by X-ray crystallography, cryo-electron microscopy, or Nuclear Magnetic Resonance (NMR) methods remains a major challenge for structural biology. While NMR can generally provide accurate 3D structures of small soluble proteins, structure determination by solution NMR of IMPs, prepared with 2H,13C,15N-isotope enrichment in stabilizing membrane-mimicking environments, can be quite challenging. Evolutionary couplings (ECs), evolution-based contact predictions derived using bioinformatics methods from multiple sequence alignments, can also provide useful information for modeling the 3D structures of IMPs. By combining EC and NMR data, we can overcome the incompleteness of NMR NOESY data obtained for perdeuterated IMP samples, and address challenges in identifying true native protein structure contacts (true positives) from the phylogenetic EC analysis. In particular, inter-helical contact information that is difficult to obtain for perdeuterated IMPs by NMR is generally well represented in the sequence co-variance EC data. The EC-NMR method will be developed using b-barrel and a-helical IMPs of known structure, and then applied to studies of IMPs of unknown structure selected from designated NIH NIAID priority pathogenic bacteria. We will (i) develop and apply the Single Protein Production (SPP) method for producing isotope-enriched IMPs in E. coli, (ii) implement a micro-scale NMR screening pipeline for IMP sample optimization, (iii) design improved algorithms for structure determination of IMPs combining ECs and NMR data, and (iv) develop standards and tools for validation of IMP structures determined by EC-NMR methods. ECs will also be combined with NMR data to identify and determine structures of multiple ?native states? of proteins. Liquid Helium (LHe) is essential for operation of the high field 500 MHZ, 600 MHz, and 800 MHz NMR systems used in this project. LHe supplies have become limited, and our delivery quotas from the vendor, Air Gas, Inc, are insufficient to maintain our superconducting NMR and MRI magnets. LHe prices have also increased 3-fold over the past few years, and the quality of LHe has been inconsistent, which can damage the cryostats. Several NIGMS- (and other NIH- and NSF-) funded projects depend on superconducting magnets maintained in the RPI Magnetic Resonance Core Facility. These NMR and MRI systems represent a significant investment by RPI and NIH, and are at risk for shutdown due to LHe cost and accessibility. Hence, it is critical that we obtain a Helium Recovery System. Multiple systems have been evaluated. Both installation and long-term maintenance plans are well- developed. The proposed LHe Recovery System will significantly reduce NMR instrument operating costs, and ensure a reliable supply of LHe cryogen which is critical for the long-term stability of this research program.